Prevention of relapse in Drug Sensitive Pulmonary Tuberculosis patients with and without Vitamin D3 supplementation: A double blinded randomized control clinical trial Sanjeev Sinha (  drsanjeevsinha@gmail.com ) All India Institute of Medical Sciences Himanshu Thukral All India Institute of Medical Sciences Imtiyaz Shareef All India Institute of Medical Sciences Devashish Desai All India Institute of Medical Sciences Binit Singh All India Institute of Medical Sciences Bimal Kumar Das All India Institute of Medical Sciences Sahajal Dhooria Post Graduate Institute of Medical Education and Research Rohit Sarin NITRD, New Delhi Rupak Singla NITRD, New Delhi Saroj Kumari Meena NITRD, New Delhi R. M. Pandey All India Institute of Medical Sciences Shivam Pandey All India Institute of Medical Sciences Digambar Behera Post Graduate Institute of Medical Education and Research Sunil Sethi Post Graduate Institute of Medical Education and Research Ashumeet Kajal Post Graduate Institute of Medical Education and Research Page 1/18 Rakesh Yadav Post Graduate Institute of Medical Education and Research Ashutosh Nath Aggarwal Post Graduate Institute of Medical Education and Research Sanjay Bhadada Post Graduate Institute of Medical Education and Research Research Article Keywords: Pulmonary Tuberculosis, Vitamin D, Clinical Trial, Placebo, Relapse Posted Date: March 18th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1331808/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/18 Abstract Background: The immunomodulatory effects of vitamin D are widely recognized and a few studies have been conducted to determine its utility in the treatment of tuberculosis, with mixed results. This study was conducted to see if vitamin D supplementation in patients with active pulmonary tuberculosis (PTB) in the Indian population contributed to sputum smear and culture conversion as well as the prevention of relapse. Methods: This randomized double-blind placebo-controlled trial was conducted in three sites in India. HIV negative participants aged 15-60 years with sputum smear positive PTB were recruited according to the Revised National Tuberculosis Control Program guidelines and were randomly assigned (1:1) to receive standard anti-tubercular treatment (ATT) with either supplemental dose of oral vitamin D3 (60,000 IU/sachet weekly for rst two months, fortnightly for next four months followed by monthly for the next 18 months) or placebo with same schedule. The primary outcome was relapse of PTB and secondary outcomes were time to conversion of sputum smear and sputum culture. Results: A total of 846 subjects were enrolled between February 1, 2017 to February 27, 2021, and randomly assigned to receive either 60,000 IU vitamin D3 (n = 424) or placebo (n = 422) along with standard ATT. Among the 697 who were cured of PTB, relapse occurred in 14 participants from the vitamin D group and 19 participants from the placebo group (hazard risk ratio 0.68, 95%CI 0.34 to 1.37, log rank p value 0.29). Similarly, no statistically signi cant difference was seen in time to sputum smear and sputum culture conversion between both groups. Five patients died each in vitamin D and placebo groups, but none of the deaths were attributable to the study intervention. Serum levels of vitamin D were signi cantly raised in the vitamin D group as compared to the placebo group, with other blood parameters not showing any signi cant difference between groups. Conclusions: The study reveals that vitamin D supplementation does not seem to have any bene cial effect in the treatment of PTB in terms to the prevention of relapse and time to sputum smear and culture conversion. Trial registration: The trial protocol was registered with the ICMR, Clinical Trial Registry – India Trial registration number: CTRI/2021/02/030977 ( date of rst registration-03/02/2021). Background Tuberculosis (TB) is a debilitating contagious disease and a major global public health problem(1). A total of 1.5 million people died from TB in 2020 (including 214,000 people with HIV). Worldwide, TB is the 13th leading cause of death and the second leading infectious killer after COVID-19 (above HIV/AIDS).Eight countries account for two thirds of worldwide deaths, with India leading the count, followed by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa.(2) Page 3/18 Vitamin D, also called cholecalciferol, is a micronutrient which is recognized primarily for its role in bone mineralization. It has also been under scrutiny lately with respect to its immuno-modulatory properties. In recent years, extensive research has been done to look for other functions of vitamin D and its related metabolites. Many epidemiological studies have shown inverse relation in the levels of vitamin D and incidence of various infectious and chronic diseases(3). The major circulating metabolite of vitamin D is 1,25-hydroxyvitamin D (1,25[OH]D), which supports innate antimicrobial immune responses, thus suggesting a potential mechanism by which adjunctive vitamin D might enhance response to antituberculosis therapy (ATT)(4). Consequently, in a country with a high TB burden such as India, there is a compelling need to investigate the potential bene ts of vitamin D supplementation along with ATT in subjects with active pulmonary TB (PTB), particularly with regards to preventing relapse after successful treatment completion and cure as well as achieving early sputum and culture conversion. If so, it can be helpful in further optimizing the management of PTB. Hitherto few studies have been conducted to investigate the effect of vitamin D in the management of PTB, albeit with small sample sizes, limited dosage of vitamin D and with analysis of limited parameters. This randomized control trial was done to assess the effect of vitamin D in patient with PTB on the time to sputum smear and culture conversion as well as the risk of relapse. Methods Study design and participants: We conducted a randomized double-blind placebo-controlled trial at three tertiary care hospitals in India (All India Institute of Medical Sciences, Delhi; National Institute of Tuberculosis and Respiratory Diseases, Delhi and Post Graduate Institute of Medical Education and Research, Chandigarh). Patients were recruited from DOTS (Directly Observed Treatment, Short Course) centres in agreement with Revised National Tuberculosis Control Program (RNTCP) guidelines. New sputum smear positive subjects in the age group of 15–60 years and willing to participate were enrolled for the study. Subjects having any major surgical or medical illness or requiring hospitalization for the same or having chronic kidney disease, Human Immunode ciency Virus(HIV) infection, BMI < 15kg/m2, diabetes mellitus, chronic alcoholism, pregnant and lactating women, or receiving any kind of vitamin D / calcium supplementation or having any disorder of bone mineral homeostasis, and those refusing consent were excluded from the study. Subjects were diagnosed as a case of PTB by sputum smear examination for acid fast bacilli (AFB), for which 2 sputum samples were collected- one early morning and the second on the spot. The sputum was treated with N-acetyl-L-cysteine/NaOH and the ensuing sediment was stained by Ziehl Neelsen method. The slides were examined under oil immersion objective, and the number of AFB observed were graded from scanty to 3 + as per the recommendations of WHO. Sputum cultures were done using the Mycobacteria Growth Indicator Tube (MGIT-960) non radiometric automated isolation system (Becton Dickinson), and drug resistance was determined using the line probe assay (LPA) method. To ensure Page 4/18 eligibility of subjects, pre-randomization investigations for hepatic and renal assessment, serology for HIV, Hepatitis B and Hepatitis C as well as fasting blood glucose were done. Other investigations like chest radiograph, electrocardiogram, complete hemogram and urine microscopy were also done. The primary outcome was rate of relapse of PTB in both arms assessed up to 24 months of follow up. Secondary outcomes included time to sputum smear and culture conversion in months. Randomization: Subjects were randomly assigned (1:1) into two groups by using block randomization via a computer program. Packets containing the two interventions were prepared in accordance with the randomization numbers and were arranged serially, and then distributed consecutively to subjects according to their entry into the trial. Both the groups received standard DOTS ATT as per RNTCP guidelines. The intervention group received supplemental dose of oral vitamin D3 (60,000 IU/sachet every week for rst two months, then every fortnight for next four months and then every month for next 18 months), and the control received only placebo with an identical schedule. The vitamin D and placebo sachets were identical in terms of their taste and physical appearance. The codes of the interventions were kept secure by an investigator who was not involved in the distribution of the packets and none of the patients, clinical and laboratory investigators were informed of the treatment assignment. The protocol was followed as per the Good Clinical Practice standards and Institutional Ethical Guidelines. The protocol was approved by DHR, ICMR and institutional ethics committee of all three centers. Written informed consent was taken from all the participants. For participants below the age of 18, written informed consent was provided by their legal guardians. The trial protocol was registered with the Clinical Trial Registry – India. Procedures: Sputum smear for AFB was taken every fortnight during the intensive phase (two months), every month during the continuation phase (four months) and once every six months thereafter till the 24th month of follow up. Samples for MGIT culture were taken every fortnight for the initial 2 months and every month thereafter till 6 months of follow up. Blood and urine investigations were conducted every fortnight till two months, then every month till six months and then every six months till the 24th month follow up. Chest radiograph was done at baseline, at the end of the intensive phase, end of treatment and every 6 months thereafter. Ultrasound abdomen done at baseline, end of treatment and at 24 months. Disease severity was graded on chest radiographs as minimal, moderately advanced, and far advanced according to the American Thoracic Society recommendations.(5) Statistical analysis: Page 5/18 As per Revised National Tuberculosis Control Program annual report (2013), 12% of PTB patients receiving category-I ATT relapsed. Assuming that vitamin D supplementation for 24 months (6 months with ATT and 18 months post ATT), would reduce the relapse by 5% by the end of two years from diagnosis. To detect a 5% reduction (12% vs 7%) in the relapse between the two arms in a two-sided test with 5% alpha error and 80% power, 539 participants in each group (as per the nQuery Advisor Version 2.0). Giving an allowance of 15% each for losses in follow-up and MDR detection after randomization, 772 participants would be required per group. Therefore we need to randomize about 1550 participants. The data was analyzed using STATA version 16.0.0. Blood, urine and radiological parameters were analyzed using parametric and non-parametric analysis. Parametric data was analyzed using Student’s ttest, while non-parametric data was analyzed using Wilcoxon rank sum test. Sputum smear and culture data was analyzed using Wilcoxon (Breslow) test for equality of survivor functions. Relapse data was analyzed using Log-rank test for equality of survivor functions in STATA. Results A total of 1996 subjects were screened from February 1, 2017 and followed up till February 27, 2021 at three tertiary care hospitals in India. Of these 1150 subjects were excluded and 846 were randomly assigned to the vitamin D group (n = 424) and placebo group (n = 422). (Figure.1) Baseline and demographic characteristics did not demonstrate any signi cant difference between the two groups. Baseline BMI is shown in Table 1, with an overall mean of 19.3 ± 3.7 kg/m2. The baseline BMI amongst the two groups did not display any statistically signi cant difference (p-0.95). A majority of the participants in both groups were de cient in Vitamin D with average value of 14.2 ± 8.7ng/ml (Normal:25–80 ng/ml). Vitamin D levels amongst the two groups also did not reveal any statistically signi cant difference (p = 0.156). Similar trends were observed for calcium levels (p = 0.951), phosphate (p = 0.335), urea (p = 0.175), creatinine (p = 0.160), iPTH (p = 0.377) and hemoglobin (p = 0.957). Analysis of the chest radiographs revealed 46% of individuals having moderately affected lungs with no signi cant difference amongst the two groups at baseline (p = 0.151). Sputum smear pro le at baseline showed 49% of subjects had 1 + bacilli (AFB), 21% had 2 + and 30% had 3+, with no signi cant difference seen among both groups (p = 0.46). Page 6/18 Table 1 Baseline and demographic characteristics Demographic Pro le ( n = 846) (MEAN ± SD) Observations Vitamin D arm (424) Placebo arm (422) Gender Male – 276(65%) Male – 279 (66%) Female – 148 (35%) Female – pvalue Overall (n-846) 143 (34%) Male – 555 (65%) Female – 291 (35%) Age (in years) 29.2 ± 11.6 29.4 ± 11.4 0.821 29.3 ± 11.5 Weight (Kg) 48.7 ± 9.5 49.1 ± 9.8 0.553 48.9 ± 9.6 Height (cm) 158.3 ± 11.1 158.9 ± 10.8 0.457 158.6 ± 10.9 BMI (Kg/m2) 19.3 ± 3.9 19.3 ± 3.6 0.955 19.3 ± 3.7 Calcium (mg/dl) 8.8 ± 0.6 8.7 ± 0.6 0.951 8.8 ± 0.7 Phosphate(mg/dl) 3.5 ± 1.0 3.4 ± 0.9 0.335 3.4 ± 1.0 25OH Vitamin D 3 (ng/ml) (3–63.5 ) 12.4 (2.6–61.1) 11.6 0.156 14.2 ± 8.7 iPTH (1–229.3 ) 17.8 (4–115.8 ) 15.4 0.377 21.3 ± 16.3 Hemoglobin (gm/dl) 11.9 ± 1.9 11.8 ± 1.8 0.957 11.9 ± 1.9 Urea (mg/dl) 24.3 ± 6.7 24.9 ± 6.5 0.175 24.6 ± 6.6 Creatinine (mg/dl) 0.7 ± 0.3 0.8 ± 0.3 0.160 0.7 ± 0.3 Chest X-ray severity Normal − 8 Normal − 6 0.151 Normal − 14 Mild – 117 Mild – 144 Mild – 261 Moderate- 202 Moderate- 194 Moderate- 396 Severe − 97 Severe − 78 Severe − 175 1 + − 212 1 + − 200 2 + − 92 2 + − 86 2 + − 178 3 + − 120 3 + − 136 3 + − 256 Sputum Smear Pro le Primary Outcome: Page 7/18 0.461 1 + − 412 For the nal analysis, we took patients who had successfully completed their six months of treatment and 18 months of follow up (697 out of 846). 149 subjects were censored of which 87 were lost to followup, 57 individuals provided insu cient sample, 3 had adverse event and 2 patients expired. There were 14 (4%) relapses in vitamin D arm and 19 (5.5%) relapses in placebo arm (n = 697 patients, n = 149 censored), which was however not statistically signi cant (p = 0.29). Bivariate Cox regression revealed a hazard ratio of 0.68 (95% CI: 0.34 to 1.37) having a log rank p-value of 0.29 (Table 2), thus implying a 32% reduction in relapses amongst individuals receiving vitamin D, which is not statistically signi cant. Upon testing for drug resistance, none of the relapsed participants were found to be harboring resistant organisms. All the relapsed patients were again initiated on ATT. Most of the individuals relapsed were male (n = 25) compared to female (n = 8). Table 2 Primary outcome of the study No. of subjects cured at 6 month No. of Relapse in Placebo arm (n343) No. of Relapse in Vitamin D arm (n354) Hazard Ratio (Bivariate Cox Regression) 95% CI PValue n-697 19 (5.5%) 14 (4%) 0.68 0.34– 1.37 0.290 Secondary Outcomes: Median time to sputum smear conversion in both groups was two weeks (95% CI 1.7 to 2.2 in vitamin D arm; 95% CI 1.7 to 2.6 in placebo arm) with hazard risk ratio of 1.06 (95% CI 0.92 to 1.23) and log rank p value 0.35 (Table 3). There was no signi cant difference observed between vitamin D and placebo groups with respect to median time to sputum smear conversion (n = 779 patients, n = 67 censored). Median time to culture conversion for both groups was four weeks (95% CI 3.6 to 4.3 vitamin D arm; 95% CI 3.7 to 4.3 placebo arm) with hazard risk ratio of 1.06 (95% CI 0.91 to 1.24) and log rank p value of 0.42. Again, there was no signi cant difference seen in the median time to culture conversion (n = 703 patients, n = 143 censored) between the two groups (Table 3). Page 8/18 Table 3 Secondary outcomes of the study Group (s) Subject(s) Median Time to Conversion (In Weeks) 95% CI Hazard Ratio (Bivariate Cox Regression) 95% CI pvalue 0.92– 1.23 0.35 0.91– 1.24 0.418 Time to Sputum smear conversion (n-779) Placebo 385 2 1.7– 2.6 1.0 Vitamin D 394 2 1.7– 2.2 1.06 Time to Culture Conversion (n- 703) Placebo 347 4 3.7– 4.3 1.0 Vitamin D 356 4 3.6– 4.3 1.06 The intervention arm saw a signi cant increase in vitamin D levels compared to the placebo arm following supplementation, which was maintained throughout the follow-up at 6th, 12th, 18th and 24th month (p-value < 0.001 at each interval). None of the subject in either group developed hypercalcemia. There was no signi cant difference observed in calcium level during follow up at 6th month (p = 0.62), 12th month (p = 0.52), 18th month (p = 0.17) and 24th month (p = 0.13). Further sub group analysis was done to see the relationship between BMI (Asian Indian Criteria and relapse rate.(6) It was observed that no relapse occurred in the BMI range of ≥ 25 while 63% (n = 21) of relapse cases were seen in BMI range < 18.5, 33% (n = 11) in BMI range 18.5–22.9 and 3% (n = 1) relapse case was seen in BMI range 23–24.9. On Cox regression analysis, hazard ratio was 0.50 (95% CI: 0.2–1.3, p-0.19) which shows more than 50% of relapse cases were observed in < 18.5 BMI but results are not statistically signi cant (p = 0.19). Safety pro le (non-serious and serious adverse events) and mortality pro le (all-cause deaths) analysis between the two groups (Table 4) showed that 10 (1.1%) patients died during the study ( ve in the vitamin D group and ve in the placebo group). No death was directly attributable to the study intervention. Excluding death, seven serious adverse events were recorded (4 in placebo and 3 in vitamin D arms). Two patients in vitamin D arm had drug induced liver injury, with 1 patient developing liver abscess, while 4 participants in the placebo arm had serious side effects (one developing ethambutol ototoxicity, one developed allergic reaction to the placebo, one had drug induced liver injury and one developed DRESS syndrome). A total of 14 adverse events were recorded, eight in the vitamin D group and six in the placebo group, all having renal calculi, with none requiring a change in medical therapy (Table 4). Page 9/18 Table 4 Adverse events pro le Vitamin D (n-424) Placebo (n-422) COX Regression pvalue Variable No. of Events Event / 100 personmonth No. of Events Event / 100 personmonth Hazard Ratio (Vitamin D vs Placebo) 95% CI Death 5 0.10 5 0.11 0.96 (0.2–3.3) 0.95 Serious Adverse Event 3 0.06 4 0.06 0.96 (0.1–4.7) 0.96 Adverse Event (Renal Calculus) 8 0.17 6 0.13 1.3 (0.4–3.7) 0.61 Hypercalcemia 0 0 0 0 NC - Hypervitaminosis D 0 0 0 0 NC - *NC indicates that the risk ratio could not be calculated due to no events in both the groups Discussion At the initiation of our study, we observed most of our patients suffering from active PTB being de cient in vitamin D with average value at 14.2 ± 8.7ng/ml (Normal:25–80 ng/ml). This is in agreement with similar studies across the globe; a study in US adults acknowledged the inverse relationship of vitamin D levels with the risk of latent TB infection (LTBI) (7) and another in Sudan connected low vitamin D Levels with increased risk of PTB (8). In view of its anti-in ammatory and anti-microbial qualities as elucidated by Coussens et al, one can make the assumption for Vitamin D de ciency to be a risk factor for the development of PTB.(9). However, a large study in Mongolia involving 8851 children revealed no bene cial effect of Vitamin D supplementation in limiting the risk of TB infection despite the Vitamin D levels reaching physiologic levels.(10) In our study, supplementation of high-dose oral vitamin D was safe and led to a substantial increase in plasma vitamin D concentrations in the intervention arm, but did not contribute signi cantly towards prevention of relapse. Although the number of relapses in the group without vitamin D supplementation was less, the difference was not statistically signi cant. Similar ndings were seen in a metaanalysis of 8 trials, where despite a signi cant increase in vitamin D levels, there was no bene cial effect of vitamin D as adjunctive treatment for TB(11). We observed relapses in our study to be more common in male subjects (n = 25) compared to female subjects (n = 8), which is concordant with the similar trend observed in the general population(12). Individuals having BMI > 25 in both the groups were observed to be have no relapse, a phenomenon which is also commonly seen in our clinical practice. A possible explanation for such a phenomenon may be due to the differential biophysical stresses in the lungs of these individuals, wherein young males having lower BMI are more predisposed to enlarging apical bullae, which are easy targets for TB reactivation. Studies in this purview also suggest the same.(13) Page 10/18 With regards to the therapeutic value of vitamin D in the treatment of TB, increase in vitamin D levels in serum as a result of supplementation did not translate to signi cant improvement in time to sputum smear and culture conversion in comparison with the placebo group. This is in agreement with similar studies investigating the relationship between vitamin D and PTB.(14) Wejse et al, did not observe any bene cial outcome of vitamin D supplementation on TB score, weight gain, sputum conversion, or mortality (15). A study in Georgia con rmed the inverse relationship between vitamin D levels and risk of TB, but failed to show enhanced sputum Mycobacterial clearance in patients receiving high dose vitamin D(16). According to Martineau et al, vitamin D did not improve time to sputum conversion in the study population, however, sputum conversion was signi cantly faster in patients with the tt genotype of the TaqI polymorphism of the Vitamin D Receptor gene(4). In contrast, Tukvadze et al, could not con rm whether the allele resulted in a faster sputum culture conversion for patients on vitamin D treatment. However, their study was signi cantly underpowered.(16)(4) Certain peers attributed the negative results in the studies of Martineau et al and Wejse et al to the low dosage of the intervention (4)(15). In order to address this limitation, as well as keeping in mind the higher prevalence of vitamin D de ciency in our country as compared to the western world,we supplemented our intervention group with high dose of Vitamin D (60,000 IU), but even this change did not translate to a faster sputum mycobacterium clearance.(17) No signi cant difference was detected in the safety pro le (non-serious and serious adverse events) and mortality pro le (all-cause deaths) between the two groups. The most reported non-serious adverse event in 14 patients out 846 was renal calculi. Adverse events and deaths were evenly distributed between the two groups. Our study has several strengths, including a large sample size (n = 846) with an appropriate period of follow up (2 years). Participants also received a substantial dose of vitamin D to avoid the possibility of false negative results as criticized by some in the earlier mentioned studies. The participants were closely followed up regularly throughout the study period and the administration of the intervention was directly observed, ensuring good compliance to medications and thus eliminating the confounding effect of noncompliance contributing to relapse. The proportion of missing outcome data was small and comparable in both the groups. Therefore, our ndings have a high degree of validity. Conclusions Vitamin D supplementation showed no bene t over placebo in preventing relapse of PTB and in reducing time to sputum smear and culture conversion. There were no major adverse reactions observed in either the vitamin D or placebo arm which could be ascribed to the intervention. Abbreviations AIIMS All India Institute of Medical Sciences Page 11/18 PTB Pulmonary Tuberculosis TB Tuberculosis ATT Anti-tubercular treatment DOTS Directly Observed Treatment, Short Course RNTCP Revised National Tuberculosis Control Program ICMR Indian Council of Medical Research DHR Department of Health Research BMI Body Mass Index. Declarations Ethics approval and consent to participate: This study has been approved by the Institute Ethics committee, All India Institute of Medical Sciences, New Delhi. Before the start of the study, all participants provided written informed consent. For participants below the age of 18, written informed consent was provided by their legal guardians. Participants were informed that they had the right to discontinue their participation or withdraw from the study at any time. We con rm that all methods were performed in accordance with the relevant guidelines and regulations (e.g., Declaration of Helsinki). Consent for publication: Not Applicable Availability of data and materials: De-identi ed participant data will be provided upon reasonable request to drsanjeevsinha@gmail.com. Competing Interests: The authors declare that they have no competing interests. Funding: Department of Health Research, Ministry of Health and Family Welfare, Government of India. Author contributions: SS conceptualized the study, determined methodology, recruited participants at AIIMS New Delhi, collated / analyzed data and wrote the rst draft of the manuscript. HT contributed to participant recruitment at AIIMS New Delhi, data analysis and manuscript writing. IS and DD contributed to data analysis and manuscript writing. Page 12/18 BS,SuS,RY and BKD performed the laboratory work and contributed to manuscript editing. SD,AK,ANA,SB and DB contributed to patient recruitment at PGIMER Chandigarh as well as analysis and editing of the manuscript. SKM, RSi and RS contributed to patient recruitment at NITRD New Delhi as well as analysis and editing of the manuscript. RMP and SP performed data analysis, creating the gures and tables and manuscript editing. SS and HT accessed and veri ed the data. All authors approved of the nal manuscript. Acknowledgements: We thank the staff members of the DOTS clinic for their help during patient enrolment and follow-up. We acknowledge the support and coordination of all staff of the project and laboratory staff of Department of Medicine and Microbiology. We thank Department of Health Research, Ministry of Health and Family Welfare, Government of India for funding this project. References 1. WHO Global Report. Geneva, Switzerland: World Health Organiser, 2014 2. WHO TB Report October 2021. Switzerland, Geneva: World Health Organisation, 2021. 3. Holick MF. Vitamin D: A millenium perspective. J Cell Biochem. 2003 Feb 1;88(2):296–307. doi: 10.1002/jcb.10338. PMID: 12520530. 4. Martineau AR. Old wine in new bottles: vitamin D in the treatment and prevention of tuberculosis. Proc Nutr Soc. 2012 Feb;71(1):84–9. doi: 10.1017/S0029665111003326. Epub 2011 Nov 29. PMID: 22123447. 5. National Tuberculosis Association, National Tuberculosis and Respiratory Disease Association. Diagnostic standards and classi cation of tuberculosis. Eleventh Ed. 1961 New York. https://catalog.hathitrust.org/Record/010152594. . Aziz N, Kallur SD, Nirmalan PK. Implications of the revised consensus body mass indices for asianindians on clinical obstetric practice. J Clin Diagn Res. 2014 May;8(5):OC01-3. doi: 10.7860/JCDR/2014/8062.4212. Epub 2014 May 15. PMID: 24995216; PMCID: PMC4080037. 7. Wang CY, Hu YL, Wang YH, Chen CH, Lai CC, Huang KL. Association between vitamin D and latent tuberculosis infection in the United States: NHANES, 2011–2012. Infect Drug Resist. 2019 Jul 22;12:2251–2257. doi: 10.2147/IDR.S213845. PMID: 31413602; PMCID: PMC6659785. . Elsa SSMS, Nour BM, Abakar AD, Omer IH, Almugadam BS. Vitamin D level and it is association with the severity of pulmonary tuberculosis in patients attended to Kosti Teaching Hospital, Sudan. AIMS Microbiol. 2020 Mar 13;6(1):65–74. doi: 10.3934/microbiol.2020004. PMID: 32226915; PMCID: PMC7099198. Page 13/18 9. Coussens AK, Martineau AR, Wilkinson RJ. Anti-In ammatory and Antimicrobial Actions of Vitamin D in Combating TB/HIV. Scienti ca (Cairo). 2014;2014:903680. doi: 10.1155/2014/903680. Epub 2014 Jul 2. PMID: 25101194; PMCID: PMC4102066. 10. Ganmaa D, Uyanga B, Zhou X, Gantsetseg G, Delgerekh B, Enkhmaa D, Khulan D, Ariunzaya S, Sumiya E, Bolortuya B, Yanjmaa J, Enkhtsetseg T, Munkhzaya A, Tunsag M, Khudyakov P, Seddon JA, Marais BJ, Batbayar O, Erdenetuya G, Amarsaikhan B, Spiegelman D, Tsolmon J, Martineau AR. Vitamin D Supplements for Prevention of Tuberculosis Infection and Disease. N Engl J Med. 2020 Jul 23;383(4):359–368. doi: 10.1056/NEJMoa1915176. PMID: 32706534; PMCID: PMC7476371. 11. Wu HX, Xiong XF, Zhu M, Wei J, Zhuo KQ, Cheng DY. Effects of vitamin D supplementation on the outcomes of patients with pulmonary tuberculosis: a systematic review and meta-analysis. BMC Pulm Med. 2018 Jun 28;18(1):108. doi: 10.1186/s12890-018-0677-6. PMID: 29954353; PMCID: PMC6025740. 12. Zong Z, Huo F, Shi J, Jing W, Ma Y, Liang Q, Jiang G, Dai G, Huang H, Pang Y. Relapse Versus Reinfection of Recurrent Tuberculosis Patients in a National Tuberculosis Specialized Hospital in Beijing, China. Front Microbiol. 2018 Aug 14;9:1858. doi: 10.3389/fmicb.2018.01858. PMID: 30154770; PMCID: PMC6102324. 13. Casha AR, Scarci M. The link between tuberculosis and body mass index. J Thorac Dis. 2017 Mar;9(3):E301-E303. doi: 10.21037/jtd.2017.03.47. PMID: 28449528; PMCID: PMC5394030. 14. Daley P, Jagannathan V, John KR, Sarojini J, Latha A, Vieth R, Suzana S, Jeyaseelan L, Christopher DJ, Smieja M, Mathai D. Adjunctive vitamin D for treatment of active tuberculosis in India: a randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2015 May;15(5):528–34. doi: 10.1016/S1473-3099(15)70053-8. Epub 2015 Apr 8. PMID: 25863562. 15. Wejse C, Gomes VF, Rabna P, Gustafson P, Aaby P, Lisse IM, Andersen PL, Glerup H, Sodemann M. Vitamin D as supplementary treatment for tuberculosis: a double-blind, randomized, placebocontrolled trial. Am J Respir Crit Care Med. 2009 May 1;179(9):843 – 50. doi: 10.1164/rccm.200804567OC. Epub 2009 Jan 29. PMID: 19179490. 1 . Tukvadze N, Sanikidze E, Kipiani M, Hebbar G, Easley KA, Shenvi N, Kempker RR, Frediani JK, Mirtskhulava V, Alvarez JA, Lomtadze N, Vashakidze L, Hao L, Del Rio C, Tangpricha V, Blumberg HM, Ziegler TR. High-dose vitamin D3 in adults with pulmonary tuberculosis: a double-blind randomized controlled trial. Am J Clin Nutr. 2015 Nov;102(5):1059–69. doi: 10.3945/ajcn.115.113886. Epub 2015 Sep 23. PMID: 26399865; PMCID: PMC4625591. 17. G R, Gupta A. Vitamin D de ciency in India: prevalence, causalities and interventions. Nutrients. 2014 Feb 21;6(2):729 – 75. doi: 10.3390/nu6020729. PMID: 24566435; PMCID: PMC3942730. 1 . Daley P, Jagannathan V, John KR, Sarojini J, Latha A, Vieth R, Suzana S, Jeyaseelan L, Christopher DJ, Smieja M, Mathai D. Adjunctive vitamin D for treatment of active tuberculosis in India: a randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2015 May;15(5):528–34. doi: 10.1016/S1473-3099(15)70053-8. Epub 2015 Apr 8. PMID: 25863562. Page 14/18 Figures Figure 1 Flow chart showing trial pro le Page 15/18 Figure 2 Kaplan Meir graph presenting time to culture conversion in both the groups Legend: *0 – Placebo, 1- Vitamin D , p-values< 0.05 Figure 2 shows Time to conversion of sputum culture in both group Vitamin D and Placebo. There is no signi cant difference in the Time to conversion of (sputum culture) MGIT with p-value is (0.418) Page 16/18 Figure 3 Kaplan Meir graph presenting time to sputum smear conversion in both the groups Legend: *0 – Placebo, 1- Vitamin D , p-values< 0.05 Figure 3 shows time to conversion of sputum smear in both vitamin D and placebo groups. There is no signi cant difference in the time to sputum smear conversion with (p value 0.358). Page 17/18 Figure 4 Kaplan Meir graph presenting time to relapse in both the groups. Legend: *0 – Placebo, 1- Vitamin D , p-values< 0.05 Figure 4 shows number of relapses in both vitamin D and placebo groups. There were more relapses in the placebo group but the difference was not statistically signi cant(p-value 0.290). Page 18/18